Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/16—Blood plasma; Blood serum
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
  • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
  • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
  • A61M1/3621—Extra-corporeal blood circuits
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M2202/00—Special media to be introduced, removed or treated
  • A61M2202/04—Liquids
  • A61M2202/0413—Blood
  • A61M2202/0415—Plasma
  • A61M2202/0417—Immunoglobulin

Definitions

• IA immunoapheresis using a column which has a specific ligand coupled thereto, as described below.
• Ig-THERASORB will refer to the column which is available from THERASORB Medizinische Systeme GmbH, Unterschleissheim/Munich, Germany. The specific Ig-THERASORB column is also described below.
• Example 1 For production of antibodies and virus inactivation, Example 1. For description of pre-columns and working columns, Example 2. For sterile purification of antibodies/protein destined to be coupled to the therapeutic column, Example 3. For preparation of sterile and pyrogen-free column matrix, Example 4. For activation of column matrix material and coupling of protein thereto, Example 5. For finishing of final column product, Example 6.
• the Ig-THERASORB column has coupled thereto pooled polyclonal antibodies raised in sheep immunized with pooled human immunoglobulin plus adjuvant.
• the coupled antibodies bind to human light chains such as lambda and kappa light chains, and thereby recognize and bind to both human IgG and IgM.
• the coupled antibodies also bind to IgG heavy chain.
• This method can also be used to increase the functioning time of a transgenic pig liver, for instance a pig liver which expresses human complement inhibitory factors.
• the method could:
• Ig-THERASORB or IgM-THERASORB columns There are several possibilities for the application of Ig-THERASORB or IgM-THERASORB columns, e.g.:
• Ex vivo liver perfusion using livers from discordant species can be achieved by immunoadsorption alone or in combination with immunosuppression and inhibition of complement activation.
• a primary separation system e.g. TPS
• TPS primary separation system
• the Ig-THERASORB columns were rinsed with 2,000 ml 0.9 % saline per column, in order to remove the storage buffer (PBS/acide).
• the duration of apheresis treatment depended on IgG concentration. Up to a plasma IgG concentration of ⁇ 400 mg/dl the loading volume was set to 260 ml plasma per column cycle. When an IgG concentration of ⁇ 400 mg/dl was reached, the loading volume was increased to 520 ml plasma per column cycle.
• the treatment scheme with 100 ml Ig-THERASORB columns and the ADA instrument is described:
• test series cell-free plasma was obtained from human blood (volume: 1 - 1,5 l) by centrifugation (10 min at 3000 rpm).
• 6 immunoapheresis cycles in test series 1-4 were performed, and 7 immunoapheresis cycles in test series 5, using Ig-THERASORB 100 columns/ADA.
• the loading volume of cycles 1-3 was 260 ml, that of cycles 4-6/7 was 520 ml plasma/cycle (total plasma amount processed: 2.34 l or 2.86 l in test series 5). The following analyses were performed.
• IL-6 interleukin-6
• TNF- ⁇ tumor necrosis factor alpha
• IFN- ⁇ interferon-gamma
• the objective of FACS analyses is to observe changes in the distribution (percentage) of lymphocyte populations during liver perfusion.
• fresh EDTA blood is incubated with FITC- or PE-labeled antibodies.
• the erythrocytes are subjected to lysis, and the leukocytes are purified by repeated washing steps with PBS.
• the so treated leukocytes are analyzed with the Becton Dickinson's FACScan, using SimulSET Software.
• the results of tests 2,3 and 4 are summarized in table 4.
• immunoapheresis alone does not cause any changes. Further investigations are required in order to be able to make statements about changes during the course of perfusion.
• the FACS results should be assessed with regard to the differential blood count. To achieve a better assessment of results, the following table 3 states the normal ranges of the parameters determined:
• test series were performed. Analogous to test series 1, cell-free plasma was obtained from human blood (blood volume 1 - 1.5 l) by centrifugation (10 min at 3000 rpm). The plasma was then processed using non-antibody-coupled sepharose material. Afterwards, it was mixed again with the blood cells and extracorporeal xenogeneic liver perfusion was performed.
• Table 6 and 7 includes the measuring values of five experiments each with the investigational and control groups.
• the results suggest that immunoapheresis with Ig-THERASORB columns are a safe and effective technology for removing anti-porcine IgG and IgM antibodies from human plasma.
• the human IgG level decreased by 95%, the IgM level by 71% and IgA level by 79%.
• the xenoreactive antibodies were reduced to 78% for human anti-pig IgG and 48% for human anti-pig IgM.
• the reduction of the immunoglobulins and xenoreactive antibodies of the control experiments with uncoupled Sepharose columns presented in table 7 results from the procedure-dependent plasma dilution or plasma loss. By improving this experimental setting for clinical use the observed dilutional effects could be further reduced.
• IL-6 interleukin-6
• TNF- ⁇ tumor necrosis factor alpha
• interferon gamma interleukin-6
• IL-6 interleukin-6
• TNF- ⁇ tumor necrosis factor alpha
• interferon gamma interleukin-6
• PE, FITC fluorescent dyes
• the leukocytes contained in them were counted using a hemacytometer.
• an aliquot of the sample to be tested was diluted 1:2 with a dye solution (crystal purple), thus triggering an erythrocyte lysis.
• 10 ⁇ l of the solution were pipetted into the hemacytometer and were investigated using a microscope with 100 x total magnification.
• the leukocyte numbers prior to and after plasma processing are practically identical. However, after 30 min of liver perfusion, only approx. 22 % of the leukocytes can be detected microscopically.
• the distribution (percentage) of the lymphocyte populations during plasma processing remained practically unchanged.
• the total number of T-cells had already multiplied by 2.7 or 2.1, respectively.
• activated T-cells had multiplied by 3.3 times.
• the CD4/CD8 ratio increased from 1.3 to 2.8.
• the percentage of B lymphocytes decreased from 5.0 or 3.3 % to 0.8 or 1.8 %, respectively.
• the percentage of natural killer cells decreased by factor 5-8.
• the transaminases GOT and GPT as well as the liver specific enzyme GLDH are also the parameters used for our model. Due to their relatively characteristic distribution in different cell compartments, their concentration in serum allows for certain conclusions concerning the degree of cellular damage. The investigations made so far suggest a significant difference between control and investigational.
• Bile production is the most significant parameter for clinical assessment of liver function. It was much higher in the investigational group than in the control group, indicating that Ig-THERASORB pre-treatment of human plasma spared the pig liver function to a great extent.
• the bromosulphtalein clearance test a dye elimination test for the evaluation of the liver's detoxification capacity, indicated a higher residual detoxification capacity of the organs in the investigational group. This is highly significant for future clinical use.
• Edema formation indicated by higher weight increase in the investigational pig livers, was an indirect sign of endothelial damage. It is understood that it may be necessary to exchange pig livers when each one shows signs of damage due to immune rejection.
• these data indicate that pre-treatment of patient plasma with Ig-THERASORB can preserve the function of an individual pig liver for up to 4 hours, and perhaps longer. The experiments were arbitrarily stopped at 4 hours. At this time point, three indicators of liver function suggested that the livers could have kept on functioning for much longer.
• An instrument can be designed to pump the blood, oxygen, and other solutions through the pig liver.
• Anti-human immunoglobulin coupled columns were used for the removal of immunoglobulin from the blood of human patients suffering from idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), vasculitis, and sensitizatiion to HLA. These procedures were part of controlled clinical trials carried out in Europe for the treatment of autoimmune patients whose conditions were refractory to conventional treatments, and patients in need of kidney transplant who had cytotoxic anti-HLA antibodies in their blood.
• ITP idiopathic thrombocytopenic purpura
• SLE systemic lupus erythematosus
• vasculitis erythematosus
• tubing system of the primary separation system was first filled with sterile 0.9% NaCl.
• Two anti-human Ig columns (Ig-THERASORB, Baxter, Immunotherapy Division, Europe) were connected with the primary separation system. All tubing connections were made under aseptic conditions.
• each column was rinsed before its first use with 5 liters sterile 0.9% NaCl solution, at a flow rate of 90-100 ml/min. For each subsequent use, it was sufficient to rinse each column with 2 liters of the sterile solution, at the same flow rate.
• the appropriate canulae were connected to the left and right cubital veins of the patient. Blood samples were taken. The connection to the blood cell separator was put in place. Anticoagulation was accomplished with either heparin or citrate (ACD-A or ACD-B) or a combination of both. When citrate was the anti-coagulant, during the first half of the procedure, the citrate was used at a dilution of 1:22 to 1:18. In the second therapy phase, the dilution utilized was 1:12 to 1:8. Hypocalcemia is known to be a common side-effect associated with the use of a different type of column (Protein-A coupled column) because the Protein A column protocol requires returning citrate buffer to the patient.
• citrate buffer is not returned to the patient.
• symptoms of hypocalcemia were monitored (paraesthesia in fingers or lips) so that the administration of anti-coagulant citrate could be diminished accordingly. There were no reports that reduction of anticoagulant was necessary.
• Calcium tablets were kept at hand to be given in case of frank hypocalcemia, but there were no reports of any necessity to administer calcium tablets.
• the blood cell separator was filled with the patient's blood.
• the blood flow rate was kept between 50-90 ml/min.
• the liquid level was maintained at about 0.8 cm over the Sepharose_ in the column.
• the cell-free plasma was directed through the tubing system over the first column. It was important to keep the flow rate even and to monitor the plasma level over the Sepharose_ in the column. A higher plasma level was undesirable, because it would have led to a higher volume burden for the patient, and plasma loss due to plasma retention in the column.
• the column was loaded with as much plasma as possible during 15 minutes. Thereafter, the plasma flow was switched to the second column, which was likewise filled with as much plasma as possible in 15 minutes.
• the volume of processed plasma per cycle was 260 ml at the beginning and then 520 ml (see table 11).
• the plasma in the first column was flushed out using sterile 0.9% NaCl at the plasma flow rate.
• One column volume of plasma was returned to the patient together with the blood cells which had been removed.
• the first column was regenerated as follows: (1) A further rinse with 50 ml 0.9 % NaCl at a flow rate of 100 ml/min; (2) Desorption of the bound immunoglobulin with one column volume of sterile 0.2 M glycine/HCl buffer, pH 2.8. The controller of the device prevented contact between this solution and the patient. The desorbed immunoglobulin was discarded. (3) Neutralization with three column volumes of sterile PBS, pH 7.4. Testing of the neutralization using pH indicator paper. (4) Rinsing out of the PBS with at least one column volume of sterile 0.9% Nacl. The column was then ready for the next round of adsorption. Then, the filling of the columns was again automatically switched.
• Plasma loss was typically low (4-8%), and no plasma replacement was required.

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• 1996
  • 1996-11-21 EP EP96118698A patent/EP0775493A3/de not_active Withdrawn

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